Field of the Invention
This invention relates to the fields of microscopic imaging of large specimens with particular emphasis on brightfield and fluorescence imaging. Applications include imaging tissue specimens, genetic microarrays, protein arrays, tissue arrays, cells and cell populations, biochips, arrays of biomolecules, detection of nanoparticles, photoluminescence imaging of semiconductor materials and devices, and many others.
Description of the Prior Art
Several technologies are used for imaging large specimens at high resolution. Tiling microscopes record an image of a small area of the specimen using a digital camera (usually a CCD camera), the specimen is moved with a computer-controlled microscope stage to image an adjacent area, an image of the adjacent area is recorded, the stage is moved again to the next area, and so on until a number of image tiles have been recorded that together cover the whole area of the specimen. Images of each area (image tiles) are recorded when the stage is stationary, after waiting long enough for vibrations from the moving stage to dissipate, and using an exposure time that is sufficient to record the fluorescence images. These image tiles can be butted together, or overlapped and stitched using computer stitching algorithms, to form one image of the entire specimen. Such images may contain tiling artifacts caused by different focus positions for adjacent tiles. For large specimens, thousands of tiles may be required to image the entire specimen, requiring many changes in focus which increase the chances of tiling artifacts.
Strip scanning instruments are often used for imaging large specimens on microscope slides. In these instruments infinity-corrected microscope optics are used, with a high Numerical Aperture (high NA) microscope objective and a tube lens of the appropriate focal length to focus an image of the specimen directly onto a CCD or CMOS linear array sensor or TDI sensor, and with the correct magnification to match the resolution of the microscope objective with the detector pixel size for maximum magnification in the digitized image {as described in “Choosing Objective Lenses: The Importance of Numerical Aperture and Magnification in Digital Optical Microscopy”, David W. Piston, Biol. Bull. 195, 1-4 (1998)}. A linear CCD detector array with 1000 or 2000 pixels is often used, and three separate linear detectors with appropriate filters to pass red, green and blue light are used for RGB brightfield imaging. A high Numerical Aperture 20× microscope objective is often used, with a 1 mm field of view. The sample is moved at constant speed in the direction perpendicular to the long dimension of the linear detector array to scan a narrow strip across a microscope slide. The entire slide can be imaged by imaging repeated strips and butting them together to create the final image. Another version of this technology uses TDI (Time Delay and Integration) array sensors which increase both sensitivity and imaging speed. In both of these instruments, exposure is varied by changing illumination intensity and/or scan speed.
Such a microscope is shown in FIG. 1 (Prior Art). A tissue specimen 100 (or other specimen to be imaged) mounted on microscope slide 101 is illuminated from below by illumination source 110. Light passing through the specimen is collected by infinity-corrected microscope objective 115 which is focused on the specimen by piezo positioner 120. The microscope objective 115 and tube lens 125 form a real image of the specimen on linear detector array 130. An image of the specimen is collected by moving the microscope slide at constant speed in scan direction 102 along the Y direction using motorized stage 105 in a direction perpendicular to the long dimension of the detector array 130, combining a sequence of equally-spaced line images from the array to construct an image of one strip across the specimen. Strips are then assembled to form a complete image of the specimen.
For brightfield imaging, most strip-scanning instruments illuminate the specimen from below, and detect the image in transmission using a sensor placed above the specimen. In brightfield, signal strength is high, and red, green and blue channels are often detected simultaneously with separate linear detector arrays to produce a colour image.
A prior art scanning microscope for fluorescence imaging is shown in FIG. 2. A tissue specimen 100 (or other specimen to be imaged) mounted on microscope slide 101 is illuminated from above by illumination source 200. In fluorescence imaging, the illumination source is usually mounted above the specimen (epifluorescence) so that the intense illumination light that passes through the specimen is not mixed with the weaker fluorescence emission from the specimen, as it would be if the illumination source were below the specimen. Several different optical combinations can be used for epifluorescence illumination—including illumination light that is injected into the microscope tube between the microscope objective and the tube lens, using a dichroic beamsplitter to reflect it down through the microscope objective and onto the specimen. In addition, a narrow wavelength band for the illumination light is chosen to match the absorption peak of the fluorophore in use. Fluorescence emitted by the specimen is collected by infinity-corrected microscope objective 115 which is focused on the specimen by piezo positioner 120. Emission filter 205 is chosen to reject light at the illumination wavelength and to pass the emission band of the fluorophore in use. The microscope objective 115 and tube lens 125 form a real image of the specimen on TDI detector array 210. An image of the specimen is collected by moving the microscope slide at constant speed in scan direction 102 along the Y direction using motorized stage 105 in a direction perpendicular to the long dimension of the detector array 210, combining a sequence of equally-spaced, time-integrated line images from the array to construct an image of one strip across the specimen. Strips are then assembled to form a complete image of the specimen. When a CCD-based TDI array is used, each line image stored in memory is the result of integrating the charge generated in all of the previous lines of the array while the scan proceeds, and thus has both increased signal/noise and amplitude (due to increased exposure time) when compared to the result if a linear array detector were used.
A description of strip scanning instruments, using either linear arrays or TDI arrays, is given in US Patent Application Publication # US2009/0141126 A1 (“Fully Automatic Rapid Microscope Slide Scanner”, by Dirk Soenksen).
When either linear arrays or TDI arrays are used for scanning a tissue specimen, focus is maintained along the scan strip by moving microscope objective 115 with piezo positioner 120. A focus map for each strip is created before scanning with measurements at several positions along the strip and focus is maintained by the piezo positioner in accordance with the focus map; or automatic focus is achieved during scanning using a separate detector or focus-measuring device. The measurement of best focus position for autofocusing a point scanner (or one using a linear array detector) was described in “Autofocusing for wide field-of-view laser scanning imaging systems”, G. Li, S. Damaskinos & A. Dixon, Scanning 28(2), 74-75 (2006). This paper describes the use of an X-Z image acquired at each of several focus points on the specimen to produce a best focus position by segmenting the X-Z image along X and calculating a best focus position for each segment. The result of a best linear fit for these focus positions is used as the line of best focus. In the Y direction, the best focus is determined by a best linear fit to focus positions calculated for various Y locations. Spatial domain intensity gradient-based solutions were found to work better than spatial frequency domain-based solutions.
If the specimen is not flat, or the specimen is tilted about the scan direction, proper focus may not be achieved across the whole width of the strip. In addition, focus at the edge of adjacent strips may be different, making it difficult to stitch image strips together to assemble a complete image of the specimen without focus mismatch at the edge of strips. These problems are made worse when magnification is increased (which decreases depth of field) and when the width of the scan strip on the specimen is increased.
Definitions
For the purposes of this patent document, a “large microscope specimen” (or “macroscopic specimen”) is defined as one that is larger than the field of view of a compound optical microscope containing a microscope objective that has the same Numerical Aperture (NA) as that of the scanner described in this document.
For the purposes of this patent document, “TDI” or “Time Delay and Integration” is defined as the method and detectors used for scanning moving objects consisting of a CCD- or CMOS-based TDI detector array and associated electronics. In a CCD-based TDI array, charge is transferred from one row of pixels in the detector array to the next in synchronism with the motion of the real image of the moving object. As the object moves, charge builds up and the result is charge integration just as if a longer exposure were used to image a stationary object. When an object position in the moving real image (and integrated charge) reaches the last row of the array, that line of pixels is read out. In operation, the image of the moving specimen is acquired one row at a time by sequentially reading out the last line of pixels on the detector. This line of pixels contains the sum of charge transferred from all previous lines of pixels collected in synchronism with the image moving across the detector. One example of such a camera is the DALSA Piranha TDI camera. In a CMOS-based TDI detector, voltage signals are transferred instead of charge.
For the purposes of this patent document, a “frame grabber” is any electronic device that captures individual, digital still frames from an analog video signal or a digital video stream or digital camera. It is often employed as a component of a computer vision system, in which video frames are captured in digital form and then displayed, stored or transmitted in raw or compressed digital form. This definition includes direct camera connections via USB, Ethernet, IEEE 1394 (“FireWire”) and other interfaces that are now practical.
For the purposes of this patent document, “depth of focus” of a microscope is defined as the range the image plane can be moved while acceptable focus is maintained, and “depth of field” is the thickness of the specimen that is sharp at a given focus level. “Depth of focus” pertains to the image space, and “depth of field” pertains to the object (or specimen) space.
For the purposes of this patent document, “fluorescence” includes single-photon and multi-photon excitation, and photoluminescence; and “specimen” includes, but is not limited to, tissue specimens, genetic microarrays, protein arrays, tissue arrays, cells and cell populations, biochips, arrays of biomolecules, plant and animal material, insects and semiconductor materials and devices. Specimens may be mounted on or contained in any kind of specimen holder.
The “scan plane” is a plane perpendicular to the optical axis of the instrument in which the specimen is moved by the moving specimen stage. When the specimen is mounted on a microscope slide, the scan plane is parallel to the surface of the microscope slide, unless the slide is tilted with respect to the moving specimen stage.
An “object plane” is a plane in the specimen (often just below the surface) that corresponds to an “image plane” on which a real image of the object plane is formed, and on which the detector is situated. An “object line” is a line in the object plane which corresponds to an “image line” (a line in the “image plane”) on which a real image of the object line is formed, and on which a linear detector is situated. The image detected by this linear detector is a “line image”.
“Dynamic tilt” is defined as tilting the detector about the scan direction in order to maintain lateral focus across the width of a scan strip during a scan, where the degree of tilt varies during the scan to maintain lateral focus.
The “scan direction” is the direction of stage motion during scanning (the Y-direction in all diagrams).